Carbon disulfide and N,N-diethyldithiocarbamate generate thiourea cross-links on erythrocyte spectrin in vivo.

CS2, a known neurotoxicant, is used in the viscose production of rayon and is also a decomposition product of N, N-diethyldithiocarbamate, a metabolic product of the drug disulfiram used in alcohol aversion therapy. Previous in vitro investigations have demonstrated the ability of CS2 to cross-link proteins through thiourea, dithiocarbamate ester, and disulfide structures. Although in vivo studies have supported protein cross-linking as both a mechanism of neurotoxicity and a potential biomarker of effect, the chemical structures responsible for CS2-mediated protein cross-linking in vivo have not been elucidated. In the present study, the structure of one type of stable protein cross-link produced on erythrocyte spectrin by CS2 in vivo is determined. Rats were exposed to 50, 500, and 800 ppm CS2 for 13 weeks by inhalation or to 3 mmol/kg N,N-diethyldithiocarbamate administered orally on alternating days for 8 weeks. Erythrocyte spectrin preparations from control and exposed rats were hydrolyzed using 6 N HCl and separated by size-exclusion chromatography. The fraction that coeluted with the synthetic deuterated lysine-lysine thiourea internal standard was derivatized with 3-[4'-[(N,N,N-trimethylamino)ethylene]phenyl] 2-isothiocyanate and analyzed by liquid chromatography tandem mass spectrometry using selected reaction monitoring detection. Lysine-lysine thiourea was detected in spectrin preparations obtained from CS2-treated rats at 500 and 800 ppm and N, N-diethyldithiocarbamate-treated rats, but not from controls. These results establish that CS2-mediated protein cross-linking occurs in vivo through the generation of Lys-Lys thiourea and that diethyldithiocarbamate can, through in vivo release of CS2, produce the same cross-linking structure. This observation supports the utility of cross-linking of peripheral proteins as a specific dosimeter of internal exposure for CS2 and provides a mechanistic explanation to account for the high-molecular-weight neurofilament protein species isolated from rats exposed to CS2 or N, N-diethyldithiocarbamate.